Studies of nonnucleoside HIV-1 reverse transcriptase inhibitors. Part 1: Design and synthesis of thiazolidenebenzenesulfonamides

Article abstract of DOI:10.1016/j.bmc.2004.08.050

A series of thiazolidenebenzenesulfonamides have been discovered as nonnucleoside reverse transcriptase inhibitors. Compound 17a exhibited the most potent inhibitory activity against the Y181C mutant reverse transcriptase (RT). The introduction of the 4-c

Full text of DOI:10.1016/j.bmc.2004.08.050

Bioorganic & Medicinal Chemistry 12 (2004) 6171–6182
Studies of nonnucleoside HIV-1 reverse transcriptase
inhibitors. Part 1: Design and synthesis of
thiazolidenebenzenesulfonamides
Naoyuki Masuda,a,* Osamu Yamamoto,a Masahiro Fujii,a Tetsuro Ohgami,a
Jiro Fujiyasu,a Toru Kontani,a Ayako Moritomo,a Masaya Orita,a Hiroyuki Kurihara,a
Hironobu Koga,a Hideaki Nakahara,a Shunji Kageyama,a Mitsuaki Ohta,a
Hiroshi Inoue,a Toshifumi Hatta,a Hiroshi Suzuki,a Kenji Sudo,a Yasuaki Shimizu,a
Eiichi Kodama,b Masao Matsuoka,b Masatoshi Fujiwara,c Tomoyuki Yokota,c
Shiro Shigetad and Masanori Babae
aInstitute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd, 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
bLaboratory of Virus Immunology, Institute for Virus Research, Kyoto University, 53 Syogoin,
Kawaramachi, Sakyo-ku, Kyoto 606-8507, Japan
cRational Drug Design Laboratories, 4-1-1, Misato, Matsukawa-Machi, Fukushima 960-1242, Japan
dDepartment of Microbiology, School of Medicine, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan
eDivision of Antiviral Chemotherapy, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences,
Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan
Received 3 August 2004; revised 31 August 2004; accepted 31 August 2004
Available online 21 September 2004
Abstract—A random high-throughput screening (HTS) program to discover novel nonnucleoside reverse transcriptase inhibitors
(NNRTIs) has been carried out with MT-4 cells against a nevirapine-resistant virus, HIV-1IIIB-R. The primary hit, a thiazolidene-
benzenesulfonamide derivative, possessed good activity. A systematic modification program examining various substituents at the
3-, 4-, and 5-positions on the thiazole ring afforded compounds with enhanced anti-HIV-1 and reverse transcriptase (RT) inhibitory
activities. These results confirm the important role ofthe substituents at these positions and the thiazolidenebenzenesulof namide
motifas a valuable lead series for the next generation NNRTIs.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
scriptase (RT) has been one ofthe main targets, and the
majority ofdrugs used clinically are RT inhibitors. RT
inhibitors can be classified into two groups: nucleoside
reverse transcriptase inhibitors (NRTIs), which act as
chain terminators to block the elongation ofthe
HIV-1 viral DNA strand, and nonnucleoside reverse
transcriptase inhibitors (NNRTIs), which directly inhi-
bit RT enzyme by binding to the allosteric site near
the polymerase active site.4
Acquired immunodeficiency syndrome (AIDS), which is
caused by the human immunodeficiency virus type 1
(HIV-1) and results in life-threatening opportunistic
infections and malignancies, has become a major world-
wide pandemic.1,2 Three million people had died from
AIDS and 40 million people were living with HIV-1 or
3
AIDS at the end of2003. From the beginning ofanti-
HIV-1 chemotherapy development, HIV-1 reverse tran-
NRTIs inhibit RT selectively but are considerably toxic
to cellular and mitochondrial DNA synthesis.5 In this
regard, NNRTIs are more specific and less toxic than
NRTIs because they do not affect the activity ofcellular
polymerases. On the other hand, during NNRTI mono-
therapy for HIV-1-infected patients6 and in vitro culture
Keywords: Thiazolidene; Benzenesulfonamide; Nonnucleoside HIV-1
reverse transcriptase inhibitor.
*
Corresponding author. Tel.: +81 298 63 6752; fax: +81 298 52
2971; e-mail: masuda.naoyuki@yamanouchi.co.jp
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.08.050

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N. Masuda et al. / Bioorg. Med. Chem. 12 (2004) 6171–6182
O
H
N
N
O
S
H
N
H
O
S
N
N
F3C
NO2
N
Cl
S
O
N
O
N
HN
O
N
N
N
H
O
N
H
O
2 (delavirdine)
3 (efavirenz)
4
1 (nevirapine)
Figure 1. Structures ofNNRTIs and lead compound 4.
experiments,7 rapid emergence ofhighly drug resistant
In this paper, we have examined the synthesis and struc-
ture–activity relationship (SAR) analysis ofthiazolid-
enebenzenesulfonamide derivatives and found that a
novel class ofNNRTIs is active against the Y181C
and K103N mutants.
viruses is often observed. In the case of nevirapine (1),
the primary cause ofdrug resistance is the substitution
oftyrosine by cysteine at the 181 position in HIV-1
RT (Y181C).8 In fact, the Y181C mutant RT is less sus-
ceptible to nevirapine in vitro than the wild-type (WT)
enzyme.9 Furthermore, the RT mutation ofthe lysine
at the 103 position to asparagine (K103N) is frequently
observed in patients who have failed in the treatment
with antiretroviral regimens containing currently avail-
able NNRTIs, such as nevirapine (1), delavirdine (2),
and efavirenz (3).10 Therefore, it is still important to find
a new lead compound that may be able to overcome the
resistance issue ofNNRTIs ( Fig. 1).
2. Chemistry
A series of4,5-dialkyl-2-aminothiazole derivatives, 6c–e
and 6g, were synthesized (Scheme 1). The a-bromination
ofketones 5a–d under thermodynamic conditions11 and
subsequent cyclization using thiourea afforded 4,5-dialk-
yl-2-aminothiazoles 6c–e and 6g. 4-Trifluoromethyl-2-
O
N
a, b
R1
R2
NH2
R1
S
R2
6c: R1 = Me, R2 = Et
6d: R1 = Me, R2 = iPr
6e: R1 = Me, R2 = Ac
6g: R1 = i-Bu, R2 = i-Pr
5a: R1 = Me, R2 = Et
5b: R1 = Me, R2 = iPr
5c: R1 = Me, R2 = Ac
5d: R1 = i-Bu, R2 = i-Pr
N
N
N
F3C
NHBoc
O
F3C
EtOOC
NHBoc
F3C
NH2
c, d
e
f
COOEt
S
S
S
F3C
HO
7
8
9
6h
N
N
N
Cl
NHBoc
Cl
Cl
NH2
NHBoc
g
h
i, j
Cl
COOH
S
S
S
MeOOC
OHC
OHC
10
11
12
13
N
N
Cl
NH2
Cl
e
NHBoc
f
S
S
HO
6j
14
Scheme 1. Reagents: (a) TMSBr, DMSO/CH3CN; (b) thiourea/EtOH; (c) thiourea/I2, i-PrOH; (d) Boc2O, DMAP/THF; (e) MeLi/THF; (f) Et3SiH/
TFA; (g) thiourea, POCl3, Ca(OH)2, NaCl/DMF; (h) Boc2O, DMAP/dioxane; (i) NaClO2, KH2PO4, 2-methyl-2-butene/t-BuOH; (j) MeOH, water
soluble carbodiimide, HOBt, DMAP/DMF.

N. Masuda et al. / Bioorg. Med. Chem. 12 (2004) 6171–6182
6173
aminothiazole 6h was synthesized from trifluoroaceto-
acetic acid ethyl ester 7 in five steps. The iodine-medi-
ated cyclization ofester 7 with thiourea in isopropanol
and the protection ofthe resultant amine with Boc anhy-
dride provided compound 8. The bis-alkylation of 8
using methyllithium provided 5-hydroxymethylethyl-2-
aminothiazole 9. The treatment of 9 with triethylsilane
and trifluoroacetic acid effectively removed the tertiary
hydroxy group and the Boc group, respectively, to af-
ford the desired 4-trifluoromethyl-2-aminothiazole 6h.
4-Chloro-2-aminothiazole 6j was synthesized from
chloroacetic acid 10 in six steps. The cyclization of
chloroacetic acid 10 with thiourea, and subsequent
formylation and chlorination, resulted in 5-formylthiaz-
ole 11.12 Treatment of 11 with Boc2O, followed by
oxidation and esterification, afforded the methyl ester
13. Treatment ofcompound 13 with methyllithium, then
triethylsilane and trifluoroacetic acid afforded the
aminothiazole 6j.
The 2-aminothiazoles 6a–j were reacted with 3-nitro-
benzenesulfonylchloride (15) to afford the thiazolidene-
benzenesulfonamide derivatives 4 and 16a–j (Scheme
2). Methylation of 4 and 16a–j using methyl iodide in
the presence ofsodium hydride afforded the thiazolid-
enesulfonamides 17a–k. Compound 4 was also con-
verted into the corresponding N-ethyl derivative 17l in
a similar manner employing ethyl iodide. Alkylation of
the nitrogen at the 3-position in 17a–l was confirmed
by NMR (Fig. 2). A nuclear Overhauser effect (NOE)
was observed between the 3-methyl protons and the 4-
methyl protons, but not between the 3-methyl protons
NH2
N
O
S
O
O
S
O
H
N
R1
b
N
N
a
O
S
O
N
NO2
NO2
Cl
+
S
R1
R1
NO2
S
S
R2
R2
R2
6a: R1 = Me, R2 = t-Bu
6b: R1 = Me, R2 = H
6c: R1 = Me, R2 = Et
6d: R1 = Me, R2 = i-Pr
6e: R1 = Me, R2 =Ac
6f: R1 = H, R2 = i-Pr
6g: R1 = i-Bu, R2 = i-Pr
6h: R1 = CF3, R2 = i-Pr
15
4: R1 = Me, R2 = t-Bu
16a: R1 = Me, R2 = H
16b: R1 = Me, R2 = Me
16c: R1 = Me, R2 = Et
16d: R1 = Me, R2 = i-Pr
16e: R1 = Me, R2 = Ac
16f: R1 = H, R2 = i-Pr
16g: R1 = i-Bu, R2 = i-Pr
16h: R1 = CF3, R2 = i-Pr
16i: R1 = COOMe, R2 = i-Pr
16j: R1 = Cl, R2 = i-Pr
17a: R1 = Me, R2 = t-Bu
17b: R1 = Me, R2 = H
17c: R1 = Me, R2 = Me
17d: R1 = Me, R2 = Et
17e: R1 = Me, R2 = i-Pr
17f: R1 = Me, R2 = Ac
17g: R1 = H, R2 = i-Pr
17h: R1 = i-Bu, R2 = i-Pr
17i: R1 = CF3, R2 = i-Pr
6i: R1 = COOMe, R2 = i-Pr
6j: R1 = Cl, R2 = i-Pr
17j: R1 = COOMe, R2 = i-Pr
17k: R1 = Cl, R2 = i-Pr
c
O
S
O
S
H
N
N
N
N
NO2
NO2
S
O
S
O
4
17l
d, e
O
S
O
S
N
N
N
N
NO2
NO2
S
O
S
O
O
17f
18
O
S
O
O
S
O
O
S
O
N
N
N
N
N
N
f, g, h
i
NO2
NO2
NO2
MeOOC
H2NOC
NC
S
S
S
17j
19
20
Scheme 2. Reagents: (a) Py; (b) MeI, NaH/THF; (c) EtI, NaH/THF; (d) NaBH4/EtOH; (e) MsCl, Et3N/CH2Cl2; (f ) NHaq/THF; (g) SOCl2; (h)
3
NH3aq/CHCl3; (i) POCl3, DMF/1,2-dichloroethane.

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N. Masuda et al. / Bioorg. Med. Chem. 12 (2004) 6171–6182
Previous studies have shown that two aromatic systems
arranged in a ꢀbutterfly-like orientationꢁ are required for
NOE
13,14
O
S
the inhibition ofRT enzymes.
We therefore at-
N
N
NO2
tempted to alkylate the ring nitrogen on the thiazole ring
ofcompound 4 in order to confer a ꢀbutterfly-like orien-
tationꢁ on the conformation of 4. The introduction of
the methyl group at the 3-position on the thiazole ring,
compound 17a, resulted in a 7- and 2.5-fold increase of
the activity against Y181C and K103N RTs, respec-
tively, when compared to unsubstituted compound 4.
In contrast, the introduction ofan ethyl group (com-
pound 17l) at the 3-position significantly reduced the
activity against the enzymes, confirming that the methyl
group at the 3-position is crucial to the inhibition of
these RTs. X-ray crystallography of 17a revealed that
the 3-methylated thiazolidenesulfonamide framework
had a (Z)-thiazolidene conformation and provided a
ꢀbutterfly-likeꢁ structure in the two aromatic systems,
as observed in other NNRTIs (Fig. 3).13,14 In the case
of 17a, one oxygen atom ofthe sulof nyl group existed
in the same plane ofthe phenyl ring, and the other ex-
isted in the plane on the thiazole ring. As a result, the
RT inhibition was enhanced by the rigid conformation
ofthe ꢀbutterfly-likeꢁ (Z)-structure derived from methyl-
ation at the 3-position on the thiazole ring. Although the
activity of 17a against the WT RT was similar to that of
4, the anti-HIV-1 activity of 17a was 6-fold stronger
than that of 4. Consequently, the TI value of 17a ex-
ceeded 290. Additionally, the introduction ofthe methyl
group may be responsible for better cell permeability or
compound stability under assay conditions.
S
O
17a
Figure 2. Confirmation ofthe structure of 17a. An NOE was observed
between the 3-methyl and 4-methyl protons on the thiazole ring,
whereas no NOE signal was observed between the 3-methyl proton on
the thiazole ring and the phenyl ring.
and aromatic protons, therefore the alkylated position
was assigned to the ring nitrogen. Treatment ofketone
17f with sodium borohydride and dehydration ofthe
resulting alcohol generated vinyl derivative 18. Hydro-
lysis of 17j and subsequent amidation afforded carbox-
amide 19, which on dehydration afforded nitrile 20.
3. Results and discussion
In order to identify the compounds that exhibit anti-
HIV-1 activity against the Y181C mutant ofHIV-1,
we conducted a large-scale high-throughput screening
(HTS) ofin-house compound libraries in MT-4 cells
acutely infected with HIV-1IIIB-R. HIV-1IIIB-R is a nevi-
rapine- and MKC-442-resistant HIV-1 strain, having
the Y181C mutation. Our screening identified com-
pound 4 as a primary hit, which possessed good anti-
HIV-1 activity against both HIV-1IIIB and HIV-1IIIB-R
with EC50 values of0.50 and 0.79 lM, respectively (data
not shown). Compound 4 inhibited WT, Y181C, and
K103N RTs with IC50 values of0.37, 0.47, and 32 lM,
respectively (Table 1).
We next investigated the effect ofthe substituents at the
5-position on the thiazole ring. As shown in Table 2,
among the compounds possessing a methyl group
at the 4-position, compounds 17a, 17c–e, and 18
retained the inhibitory activity against the WT RT.
However, the introduction ofan acetyl group at the 5-
position (compound 17f) led to a dramatic loss ofthe
activity. Substitution with bulkier alkyl groups such as
tert-butyl and isopropyl (compounds 17a and 17e; IC50
values of0.066 and 0.071 lM, respectively) exhibited a
superior activity against the Y181C RT but not against
the WT or K103N RT: therefore the activity against the
In order to increase the inhibitory activity against WT,
Y181C, and K103N RTs as well as HIV-1 replication,
we synthesized a series ofcompound 4 analogues and
evaluated for their inhibitory effects on these enzymes
and HIV-1 replication in acutely infected MT-4 cells
(Tables 1 and 2).
Table 1. In vitro activity of3-substituted thiazolidene derivatives
R
N
O
S
N
NO2
S
O
Compounds
R
IC50 (lM)a
EC50 (lM)b
CC50 (lM)c
TId
WT
K103N
Y181C
4
H
0.37
0.27
6.0
32
13
0.47
0.066
0.12
0.50
0.085
>25
>25
11.5
>50
>290
<1
17a
17l
Me
Et
>50
>11.5
a Compound concentration required to achieve 50% inhibition ofrecombinant HIV-1 RT activity.
b Compound concentration required to achieve 50% protection ofMT-4 cells from the HIV-1 induced CPE as determined by the MTT method.
c Compound concentration required to reduce the viability ofmock-infected MT-4 cells as determined by the MTT method.
d Therapeutic index (CC50/EC50).

N. Masuda et al. / Bioorg. Med. Chem. 12 (2004) 6171–6182
Table 2. In vitro activity of4- and 5-substituted thiazolidene derivatives
6175
O
S
N
N
NO2
R1
S
O
R2
Compounds
R1
R2
IC50 (lM)a
EC50 (lM)b
CC50 (lM)c
TId
WT
0.37
0.27
>50
K103N
Y181C
4
17a
32
13
0.47
0.066
>50
33
0.50
0.085
>25
>25
>25
>25
>25
>25
>25
>25
>25
21.4
>25
>25
24
>50
>290
—
Me
Me
t-Bu
H
17b
17c
>50
>50
6.3
Me
Me
Me
Et
0.97
0.85
0.34
1.1
0.23
>23
>110
>250
—
17d
17e
0.47
0.071
>50
0.70
>25
0.26
>25
0.13
9.6
Me
Me
i-Pr
Ac
20
0.10
17f
17g
>50
0.60
>50
0.60
>50
>50
>50
>50
42
>25
H
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
CH@CH2
i-Pr
i-Pr
3.1
>8
17h
17i
i-Bu
CF3
COOMe
Cl
>21.4
0.26
>25
—
>96
—
17j
17k
>50
6.9
6.2
0.077
0.15
0.048
0.097
>25
500
45
18
19
Me
CONH2
CN
4.4
39
0.62
>50
>50
1.1
>25
0.64
>1.9
7.5
>25
>25
>25
>25
8.5
—
20
0.71
>35
>4700
>6400
3200
Nevirapine (1)
Delavirdine (2)
Efavirenz (3)
0.0026
0.042
0.0069
0.0053
0.0039
0.0027
4.8
0.021
0.0040
a–dSee footnotes in Table 1.
Figure 3. X-ray crystallography ofcompound 17a.
Figure 4. Docking study of 17a within RT nonnucleoside binding site.
Y181C RT is affected by the nature of5-position substit-
uents. These results also suggest that the alkyl group at
the 5-position exists near the Y181 ofthe HIV-1 RT. In
the docking study of 17a with a RT nonnucleoside bind-
ing site, it was predicted that the tert-butyl group would
occupy the hydrophobic pocket constructed by Y181,
Y188, and W229 and that it was positioned near the
Y181 (Fig. 4). Therefore, the bulky alkyl groups at the
5-position on the thiazole ring seem to be crucial for
the inhibition ofthe Y181C RT. With respect to the sub-
stituents at the 5-position on the thiazole ring, both
ethyl (17d) and vinyl (18) groups improved the inhibi-
tory activity against the K103N RT, and 17d and 18
were significantly more potent than 4. The K103N
mutation reduced sensitivity to nevirapine and delavir-
dine 423- and 114-fold, respectively, but compounds
17a, 17d, and 18 were only 48-, 7-, and 41-fold less
potent against the K103N mutant, respectively. Com-
pounds 17a, 17d, 17e, and 18 showed potent anti-HIV-
1 activity with EC50 values of0.085, 0.23, 0.10, and
0.097lM, respectively. For each compound, its EC50 va-
lue was strongly associated with its IC50 value for WT
RT.
In order to find more potent inhibitors, further modifi-
cations at the 4-position on the thiazole ring were per-
formed. Among the 5-isopropyl derivatives prepared,

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N. Masuda et al. / Bioorg. Med. Chem. 12 (2004) 6171–6182
those with sterically small substituents at the 4-position
exhibited the strong activity against both ofthe WT and
the Y181C RTs: no apparent preference for either elec-
tron-withdrawing or electron-donating substituents was
observed. Those compounds with methyl (17e), trifluoro-
methyl (17i), and cyano (20) substituents at the 4-posi-
tion possessed the inhibitory activity similar to that of
the unsubstituted compound (17g). However, the com-
pounds with a bulkier iso-butyl group (17h) or hydro-
philic groups, such as methoxycarbonyl (17j) and
carbamoyl (19), showed a lack ofRT inhibition. The
4-methyl derivative (17e) showed the strongest activity
against the Y181C RT with an IC50 value of0.071 lM,
which was five times greater than its activity against
the WT RT (IC50: 0.34lM). In contrast to 17e, the 4-
chloro derivative (17k) showed a stronger activity
against the WT RT with an IC50 value of0.077 lM,
and this value was about twice stronger than against
the Y181C RT (IC50: 0.13lM). The introduction ofa
chloro group to the 4-position (17k) also improved the
activity against K103N RT (IC50: 6.9lM) when com-
pared to 17e. Among the 5-isopropyl derivatives, tri-
fluoromethyl (17i), chloro (17k), and cyano derivatives
(20) possessed potent anti-HIV-1 activity with EC50 val-
ues of0.26, 0.048, and 0.71 lM, respectively. The SAR
oftheir anti-HIV-1 activity also correlated well with
the SAR deduced from the inhibitory activity against
WT RT.
benzenesulfonamide derivatives indicates that the ꢀbut-
terfly-likeꢁ conformation derived from the sulfonamide
structure is important for the activity against the WT
RT and that the methyl substituent at the 3-position
on the thiazole ring is crucial for the fixed configuration
to enhance the activity. The inclusion ofhydrophobic
bulky substituents at the 5-position on the thiazole ring
is favorable for enhanced activity against the Y181C
RT. Among the newly synthesized compounds, both
17a and 17k potently inhibit HIV-1 replication with
EC50 values of0.085 and 0.048 lM, respectively. They
also have a potent activity against the WT and Y181C
RT and to a lesser extent against the K103N RT. The
thiazolidenebenzenesulfonamide analogues provide val-
uable leads for the discovery of the next generation
NNRTIs.
5. Experimental
5.1. Chemistry
Melting points were determined on a Yanaco micro-
melting apparatus or Buchi B-545 melting point appara-
¨
tus and are uncorrected. Proton magnetic resonance (1H
NMR) spectra were obtained in CDCl3 or dimethylsulf-
oxide-d6 (DMSO-d6) using a JEOL JNM-EX90, JNM-
EX400, JNM-GX500, or JNM-A500 spectrometer.
Chemical shifts were expressed in d (ppm) values with
tetramethylsilane as an internal standard (in NMR
description, s: singlet, d: doublet, t: triplet, m: multiplet,
br: broad peak). Mass spectra (MS) were recorded on a
JEOL JMS-DX300 or a HITACHI M-80 mass spec-
trometer. Elemental analysis was carried out on Yanaco
MT-3 or MT-5 CHN analyzer and a Yokogawa
IC7000S Ion Chromatoanalyzer. Chromatographic sep-
arations were performed using a silica gel column
(Merck Kieselgel 60). Analytical thin-layer chromato-
graphy (TLC) was carried out on precoated glass plates
(Merck Kieselgel 60F254). The structure oftarget com-
Taken together, three factors affect the activity against
HIV-1 RT in this series. First, the ꢀbutterfly-likeꢁ struc-
ture ofthe thiazolidenebenzenesulofnamide is crucial
for RT inhibition. Second, the inclusion of bulky sub-
stituents, such as tert-butyl and isopropyl, at the 5-posi-
tion on the thiazole ring enhances the inhibitory activity
against the Y181C RT. Third, substituents with an
appropriate size at the 4-position on the thiazole ring,
such as methyl and chloro groups, are necessary for
RT inhibition.
1
Since there was a good correlation between the EC50
values for HIV-1 replication and the IC50 values for
the WT RT, we concluded that compound 4 and its re-
lated compounds were NNRTIs. Among them, the 5-
tert-butyl-4-methyl (17a) and 4-chloro-5-isopropyl ana-
logues (17k) showed the most potent anti-HIV-1 activity
with EC50 values of0.085 and 0.048 lM, respectively,
which represent 6–10-fold improvements over the pri-
mary hit compound 4, while still retaining good TI
values.
pounds were deduced from mass spectral and H NMR
data, and were verified by a single crystal X-ray struc-
ture determination for compound 17a (Fig. 3).
The following known materials were prepared as de-
scribed in the literature: 6a,15 6f,16 6i,17 or obtained from
commercial suppliers: 6b, 15.
5.1.1. 5-Ethyl-4-methylthiazole-2-amine (6c). To a stirred
solution of2-butanone ( 5a, 5.00g, 58.1mmol) and
bromotrimethylsilane (8.3mL, 63.9mmol) in acetonitrile
(50mL) was added slowly dropwise dimethylsulfoxide
(4.5mL, 63.9mmol) under ice-bath cooling. After stir-
ring for 15min, the solution was warmed to room tem-
perature and stirred for 2h. The reaction mixture was
poured into ice water and extracted with diethyl ether.
The organic layer was washed with brine, then dried
over anhydrous sodium sulfate, and evaporated under
reduced pressure. Ethanol (50mL) and thiourea
(4.42g, 58.1mmol) was added to the residue and ref-
luxed for 1h. The mixture was cooled to room tempera-
ture, and the resulting precipitate was collected by
filtration. The precipitate was washed with diethyl ether
These results present a significant springboard for the
further improvement ofthe activity spectrum ofthiazo-
lidenebenzenesulfonamide analogues as novel NNRTIs.
4. Conclusion
The HTS ofan in-house compound library in MT-4 cells
acutely infected with HIV-1IIIB-R has been successfully
applied to the discovery ofa novel class ofNNRTIs,
which have potent inhibitory activity against both WT
and Y181C RT. The SAR analysis ofthese thiazolidene-

N. Masuda et al. / Bioorg. Med. Chem. 12 (2004) 6171–6182
6177
and hexane to give 6c hydrobromide (2.10g, 15%) as a
pale yellow solid. H NMR (DMSO-d6) d: 1.10 (3H, t,
J = 7.5Hz, CH3 of t-Bu), 2.11 (3H, s, CH3 ofEt), 2.58
5.1.7. 5-Isopropyl-4-trifluoromethyl-1,3-thiazol-2-amine
(6h). To a stirred solution of 9 (2.97g, 9.11mmol) in tri-
fluoroacetic acid (40mL) was added triethylsilane
(4.35mL, 27.3mmol) and stirred at room temperature
for 2h. The reaction mixture was concentrated under re-
duced pressure and the residue was purified by silica gel
column chromatography (ethyl acetate–hexane) to give
6h (2.20g, quantitative) as colorless syrup. 1H NMR
(CDCl3) d: 1.30 (6H, d, J = 6.8Hz, CH3 of i-Pr), 3.46
(1H, heptet, J = 6.8Hz, CH of i-Pr), 7.06 (2H, br s,
NH2); FAB-MS m/z: 211 (M++1).
1
(2H, q, J = 7.5Hz, CH2 ofEt), 9.15 (2H, br s, NH );
2
FAB-MS m/z: 143 (M++1).
The following compounds were obtained in the same
manner.
5.1.2. 4-Methyl-5-isopropylthiazole-2-amine (6d). 57%
yield; 1H NMR (CDCl3) d: 1.11 (6H, d, J = 6.8Hz,
CH3 of i-Pr), 1.97 (3H, s, 4-Me), 3.02 (1H, heptet,
J = 6.8Hz, CH of i-Pr), 6.49 (2H, br s, NH2); FAB-
MS m/z: 157 (M++1).
5.1.8. 2-Amino-4-chloro-1,3-thiazole-5-carbaldehyde (11).
To a stirred solution ofthiourea (100g, 1.31mol) in
N,N-dimethylformamide (1.0L) was added portionwise
chloroacetic acid (124g, 1.31mol) and stirred at 40ꢁC
for 2h. To the stirred reaction mixture under ice-bath
cooling was added dropwise phosphorous oxychloride
(424mL, 4.59mol) for 1h. The mixture was stirred at
60ꢁC for 30min, and then stirred at 90ꢁC for 5h. The
reaction mixture was poured into ice-water and sodium
chloride (456g, 10.2mol), calcium hydroxide (336g,
5.93mol) was added. The resulting precipitate was col-
lected by filtration and washed with water until the fil-
trate was neutral. The precipitate was dried under
reduced pressure to give 11 (165g, 72%) as pink solid.
5.1.3.
1-(2-Amino-4-methyl-1,3-thiazol-5-yl)ethanone
1
(6e). 84% yield; H NMR (DMSO-d6) d: 2.44 (3H, s,
CH3 ofAc), 2.52 (3H, s, 4-Me), 9.80 (2H, br s, NH );
2
FAB-MS m/z: 157 (M++1).
5.1.4. 4-Isobutyl-5-isopropylthiazole-2-amine (6g). 44%
1
yield; H NMR (DMSO-d6) d: 0.85 (6H, d, J = 6.8Hz,
CH3 of i-Bu), 1.12 (6H, d, J = 6.8Hz, CH3 of i-Pr),
1.89 (1H, m, CH of i-Bu), 2.19 (2H, d, J = 6.8Hz,
CH2 of i-Bu), 3.03 (1H, heptet, J = 6.8Hz, CH of i-
Pr), 6.50 (2H, br s, NH2); FAB-MS m/z: 199 (M++1).
1
Two tautomers were observed on H NMR spectrum.
5.1.5. Ethyl 2-[(tert-butoxycarbonyl)amino]-4-trifluoro-
methyl-1,3-thiazole-5-carboxylate (8). To a stirred mix-
ture ofethyl trifluoroacetoacetate (7.36g, 60mmol),
thiourea (13.68g, 180mmol), and isopropanol (30mL)
was added iodine (11.42g, 90.0mmol) and the mixture
was refluxed for 3h. The reaction mixture was concen-
trated under reduced pressure, and the residue was
recrystallized from methanol–water. To a tetrahydrofu-
ran (70mL) solution ofthe precipitate, di- tert-butyl
dicarbonate (7.61g, 34.9mmol), and N,N-dimethyl-
aminopyridine (178mg, 1.46mmol) was added. Then
the mixture was stirred at 60ꢁC for 1h. The reaction
mixture was concentrated under reduced pressure, and
the residue was purified by silica gel column chromato-
graphy (ethyl acetate–toluene) to give 7 (10.43g, 49%) as
1H NMR (DMSO-d6) one tautomer: d: 8.70 (1H, s,
NH2), 9.92 (1H, s, CHO), 13.20 (1H, br s, NH2) and
the other tautomer: 8.74 (2H, s, NH2), 9.64 (1H, s,
CHO); FAB-MS m/z: 237 (M++1).
5.1.9. tert-Butyl (4-chloro-5-formyl-1,3-thiazol-2-yl)carb-
amate (12). To a stirred solution of 11 (34.4g, 0.212mol)
in 1,4-dioxane (500mL) was added di-tert-butyl dicarbo-
nate (55.4g, 0.254mol) and N,N-dimethylaminopyridine
(2.58g, 0.021mol). The reaction mixture was stirred at
60ꢁC for 2h. The solvent was removed under reduced
pressure and the residue was dissolved in ethyl acetate.
The solution was washed with 5% aqueous potassium
hydrogen sulfate solution and brine, and then dried over
anhydrous sodium sulfate. The solvent was evaporated
under reduced pressure to give 12 (48.2g, 87%) as a
1
a colorless solid. H NMR (CDCl3) d: 1.55 (9H, s, CH3
of t-Bu), 1.57 (3H, t, J = 7.1Hz, CH3 ofEt), 4.36 (2H, q,
1
brown solid. H NMR (DMSO-d6) d: 1.50 (9H, s, CH3
of t-Bu), 9.86 (1H, s, CHO), 12.53 (1H, s, NH);
J = 7.1Hz, CH2 ofEt), 8.48 (2H, br s, NH ); FAB-MS
2
m/z: 341 (M++1).
FAB-MS m/z: 263 (M++1).
5.1.6. tert-Butyl [5-(1-hydroxy-1-methylethyl)-4-trifluoro-
methyl-1,3-thiazol-2-yl]carbamate (9). Under argon
atmosphere, to a tetrahydrofuran (200mL) solution of
8 (4.25g, 12.5mmol) was added dropwise 1.14M methyl-
lithium diethyl ether solution (44mL, 50mmol) at
ꢀ78ꢁC and the solution was stirred at the same temper-
ature for 0.5h. The reaction mixture was quenched with
saturated aqueous ammonium chloride solution and ex-
tracted with ethyl acetate. The organic layer was washed
with brine, and then dried over anhydrous sodium sul-
fate. The solvent was removed under reduced pressure
and the residue was purified by silica gel column chro-
matography (ethyl acetate–hexane) to give 9 (2.97g,
73%) as a colorless solid. 1H NMR (CDCl3) d: 1.53
(9H, s, CH3 of t-Bu), 1.71 (6H, s, Me), 2.43 (1H, s,
OH), 8.36 (1H, br s, NH); FAB-MS m/z: 327 (M++1).
5.1.10. Methyl 2-[(tert-butoxycarbonyl)amino]-4-chloro-
1,3-thiazole-5-carboxylate (13). A stirred mixture of 12
(30.0g, 114mmol), potassium dihydrogensulfate
(46.5g, 342mmol) solution in water (200mL) and 2-
methyl-2-butene (157mL, 1.48mol) in tert-butanol
(1.20L) was added dropwise sodium chlorite (61.9g,
684mmol) in water (120mL) under ice-bath cooling.
The mixture was stirred at room temperature for 6h.
To the reaction mixture, ethyl acetate was added and
washed with 5% aqueous potassium hydrogen sulfate
solution. The organic layer was alkalinized with 1M
aqueous sodium hydroxide solution and washed with
ethyl acetate. The aqueous layer was acidified to pH3
with potassium hydrogen sulfate and extracted with
chloroform. The organic layer was dried over sodium
sulfate and evaporated. The residue was dissolved in

6178
N. Masuda et al. / Bioorg. Med. Chem. 12 (2004) 6171–6182
methanol (150mL) and N,N-dimethylformamide
(300mL). To the solution, 1-hydroxybenzotriazole
(23.1g, 171mmol), 1-[3-(dimethylamino)propyl]-3-ethyl-
carbodiimide hydrochloride (54.6g, 285mmol), and
N,N-dimethylaminopyridine (134mg, 1.1mmol) was
added and stirred at room temperature for 10h. The
solvent was removed under reduced pressure and 10%
aqueous citric acid solution was added. The mixture
was extracted with ethyl acetate–toluene and washed
with water, saturated aqueous sodium hydrogen
carbonate solution and brine. The organic layer was
dried over sodium sulfate and evaporated to give
13 (19.0g, 57%) as a pale yellow solid. 1H NMR
(DMSO-d6) d: 1.50 (9H, s, CH3 of t-Bu), 3.79 (3H, s,
CH3 ofCOOMe), 12.30 (1H, s, NH); FAB-MS m/z:
293 (M++1).
5.1.14. N-(4-Methyl-1,3-thiazol-2-yl)-3-nitrobenzene-
sulfonamide (16a). 60% yield; H NMR (DMSO-d6) d:
2.10 (3H, s, Me), 6.48 (1H, s, thiazole), 7.85 (1H, t,
J = 7.8Hz, benzene), 8.22 (1H, ddd, J = 1.0, 2.0,
7.8Hz, benzene), 8.43 (1H, ddd, J = 1.0, 1.5, 7.8Hz,
benzene), 8.51 (1H, dd, J = 1.5, 2.0Hz, benzene), 12.88
(1H, br s, NH); FAB-MS m/z: 300 (M++1).
1
5.1.15. N-(4,5-Dimethyl-1,3-thiazol-2-yl)-3-nitrobenzene-
1
sulfonamide (16b). 60% yield; H NMR (DMSO-d6) d:
2.01 (3H, s, 4-Me), 2.10 (3H, s, 5-Me), 7.85 (1H, t,
J = 7.8Hz, benzene), 8.20 (1H, br d, J = 7.8Hz, benz-
ene), 8.42 (1H, ddd, J = 1.0, 2.0, 7.8Hz, benzene), 8.46
(1H, t, J = 2.0Hz, benzene), 12.63 (1H, br s, NH);
FAB-MS m/z: 314 (M++1).
5.1.16. N-(5-Ethyl-4-methyl-1,3-thiazol-2-yl)-3-nitrobenz-
enesulfonamide (16c). 11% yield; H NMR (DMSO-d6)
1
5.1.11. tert-Butyl [4-chloro-5-(1-hydroxy-1-methylethyl)-
1,3-thiazol-2-yl]carbamate (14). Compound 14 was ob-
tained from 13 in the same manner as described in the
d: 1.09 (3H, t, J = 7.7Hz, CH3 ofEt), 2.03 (3H, s,
4-Me), 2.50 (2H, q, J = 7.7Hz, CH2 ofEt), 7.83 (1H,
m, benzene), 8.22 (1H, dt, J = 1.5, 8.0Hz, benzene),
8.42 (1H, m, benzene), 8.46 (1H, m, NH); FAB-MS
m/z: 328 (M++1).
1
synthesis of 9. 98% yield as a colorless solid. H NMR
(DMSO-d6) d: 1.47 (9H, s, CH3 of t-Bu), 1.53 (6H, s,
Me), 5.82 (1H, s, OH), 11.41 (1H, s, NH); FAB-MS
m/z: 293 (M++1).
5.1.17. N-(5-Isopropyl-4-methyl-1,3-thiazol-2-yl)-3-nitro-
benzenesulfonamide (16d). 32% yield; 1H NMR (DMSO-
d6) d: 1.14 (6H, d, J = 6.8Hz, CH3 of i-Pr), 2.04 (3H, s,
4-Me), 3.10 (1H, heptet, J = 6.8Hz, CH of i-Pr), 7.85
(1H, dt, J = 0.9, 8.1Hz, benzene), 8.22 (1H, dt, J = 1.4,
7.7Hz, benzene), 8.43 (1H, m, benzene), 8.46 (1H, m,
NH); FAB-MS m/z: 342 (M++1).
5.1.12. 4-Chloro-5-isopropyl-1,3-thiazol-2-amine (6j). To
a stirred solution of 14 (20g, 6.82mmol) in trifluoroace-
tic acid (20mL) was added triethylsilane (10mL) and
stirred at room temperature for 1h. The mixture was
evaporated, saturated aqueous sodium hydrogen car-
bonate solution was added and extracted with ethyl ace-
tate. The organic layer was dried over sodium sulfate
and evaporated. The residue was purified by silica gel
column chromatography (ethyl acetate–hexane) to give
5.1.18. N-(5-Acetyl-4-methyl-1,3-thiazol-2(3H)-ylidene)-
3-nitrobenzenesulfonamide (16e). 90% yield; 1H NMR
(DMSO-d6) d: 2.44 (3H, s, CH3 ofAc), 2.46 (3H, s, 4-
Me), 7.89 (1H, t, J = 8.1Hz, benzene), 8.27 (1H, dt,
J = 1.4, 8.1Hz, benzene), 8.31 (1H, s, NH), 8.47 (1H,
m, benzene); FAB-MS m/z: 342 (M++1).
1
6j (1.17g, 97%) as a red solid. H NMR (DMSO-d6) d:
1.14 (6H, d, J = 6.9Hz, CH3 of i-Pr), 3.05 (1H, heptet,
J = 6.9Hz, CH of i-Pr), 7.20 (2H, br s, NH2); FAB-
MS m/z: 177 (M++1).
5.1.13. N-(5-tert-Butyl-4-methyl-1,3-thiazol-2-yl)-3-nitro-
benzenesulfonamide (4). To a solution of 6a (5.88g,
34.6mmol) in pyridine (200mL) was added 15 (9.46g,
42.7mmol) and the solution was stirred at room temper-
ature for 12h. The reaction mixture was poured into
water and extracted with chloroform. The organic layer
was washed with saturated aqueous sodium hydrogen
carbonate solution, 1M hydrochloric acid, and brine
and dried over anhydrous sodium sulfate. The solvent
was removed under reduced pressure. The residue was
purified by silica gel column chromatography (metha-
nol–chloroform) and recrystallized from methanol to
give 4 (8.49g, 69%) as a yellow crystals. Mp 212–
213ꢁC. 1H NMR (CDCl3) d: 1.30 (9H, s, CH3 of t-
Bu), 2.16 (3H, s, 4-Me), 7.86 (1H, t, J = 8.3Hz, ben-
zene), 8.21 (1H, dd, J = 2.0, 8.3Hz, benzene), 8.42
(1H, ddd, J = 1.8, 2.0, 8.3Hz, benzene), 8.46 (1H, t,
J = 1.8Hz, benzene), 12.58 (1H, br s, NH); FAB-MS
m/z: 356 (M++1). Anal. Calcd for C14H17N3O4S2: C,
47.31; H, 4.82; N, 11.82; S, 18.04. Found: C, 47.06; H,
4.84; N, 11.80; S, 17.84.
5.1.19. N-(5-Isopropyl-1,3-thiazol-2-yl)-3-nitrobenzene-
sulfonamide (16f). 51% yield; H NMR (DMSO-d6) d:
1
1.19 (6H, d, J = 6.8Hz, CH3 of i-Pr), 2.94 (1H, heptet,
J = 6.8Hz, CH of i-Pr), 7.05 (1H, s, thiazole), 7.86
(1H, dd, J = 7.8, 8.3Hz, benzene), 8.23 (1H, br d,
J = 7.8Hz, benzene), 8.42 (1H, ddd, J = 1.5, 1.9,
8.3Hz, benzene), 8.47 (1H, dd, J = 1.5, 1.9Hz, benzene),
12.71 (1H, br s, NH); FAB-MS m/z: 328 (M++1).
5.1.20.
N-(4-Isobutyl-5-isopropyl-1,3-thiazol-2-yl)-3-
nitrobenzenesulfonamide (16g). 85% yield; 1H NMR
(DMSO-d6) d: 0.84 (6H, d, J = 6.3Hz, CH3 of i-Bu),
1.16 (6H, d, J = 6.8Hz, CH3 of i-Pr), 1.84 (1H, m, CH
of i-Bu), 2.30 (2H, d, J = 7.3Hz, CH2 of i-Bu), 3.12
(1H, heptet, J = 6.8Hz, CH of i-Pr), 7.86 (1H, t,
J = 7.8Hz, benzene), 8.22 (1H, br d, J = 7.8Hz, ben-
zene), 8.43 (1H, br d, J = 7.8Hz, benzene), 8.48 (1H, t,
J = 2.0Hz, benzene), 12.60 (1H, br s, NH); FAB-MS
m/z: 384 (M++1).
5.1.21.
N-(5-Isopropyl-4-trifluoromethyl-1,3-thiazol-2-
yl)-3-nitrobenzenesulfonamide (16h). 78% yield; 1H
NMR (CDCl3) d: 1.14 (6H, d, J = 6.0Hz, CH3 of i-
Pr), 3.24 (1H, m, CH of i-Pr), 7.71 (1H, dt, J = 1.1,
The following compounds were obtained in the same
manner.

N. Masuda et al. / Bioorg. Med. Chem. 12 (2004) 6171–6182
6179
7.1Hz, benzene), 8.13 (1H, dd, J = 1.1, 7.1Hz, benzene),
8.26 (1H, dd, J = 1.1, 7.1Hz, benzene), 8.49 (1H, d,
J = 1.8Hz, benzene); FAB-MS m/z: 393 (M+ꢀ1).
2.0, 8.3Hz, benzene), 8.48 (1H, t, J = 2.0Hz, benzene);
FAB-MS m/z: 328 (M++1). Anal. Calcd for
C12H13N3O4S2: C, 44.03; H, 4.00; N, 12.84; S, 19.59.
Found: C, 43.84; H, 3.90; N, 12.84; S, 19.49.
5.1.22. Methyl 5-isopropyl-2-{[(3-nitrophenyl)sulfonyl]-
amino}-1,3-thiazole-4-carboxylate (16i). 75% yield;
1H NMR (CDCl3) d: 1.30 (6H, d, J = 7.0Hz, CH3 of
i-Pr), 3.95 (1H, heptet, J = 7.0Hz, CH of i-Pr), 3.93
(3H, s, CH3 ofCOOMe), 7.69 (1H, dt, J = 7.7, 8.3Hz,
benzene), 8.27 (1H, ddd, J = 1.1, 1.8, 7.7Hz, benzene),
8.38 (1H, ddd, J = 1.1, 2.4, 8.3Hz, benzene), 8.77 (1H,
t, J = 1.8Hz, benzene); FAB-MS m/z: 386 (M++1).
5.1.27. N-(5-Ethyl-3,4-dimethyl-1,3-thiazol-2(3H)-ylid-
ene)-3-nitrobenzenesulfonamide (17d). 54% yield; mp
159–160ꢁC (ethanol). 1H NMR (DMSO-d6) d: 1.09
(3H, t, J = 7.3Hz, CH3 ofEt), 2.15 (3H, s, 4-Me), 2.61
(1H, q, J = 7.3Hz, CH2 ofEt), 3.43 (3H, s, 3-Me),
7.85 (1H, dd, J = 7.8, 8.3Hz, benzene), 8.25 (1H, ddd,
J = 1.0, 1.9, 7.8Hz, benzene), 8.44 (1H, ddd, J = 1.0,
1.9, 8.3Hz, benzene), 8.49 (1H, t, J = 1.9Hz, benzene);
FAB-MS m/z: 342 (M++1). Anal. Calcd for
C13H15N3O4S2: C, 45.73; H, 4.43; N, 12.31; S, 18.78.
Found: C, 45.78; H, 4.48; N, 12.45; S, 18.76.
5.1.23. N-(4-Chloro-5-isopropyl-1,3-thiazol-2-yl)-3-nitro-
benzenesulfonamide (16j). 35% yield; H NMR (DMSO-
1
d6) d: 1.21 (6H, d, J = 6.8Hz, CH3 of i-Pr), 3.12 (1H,
heptet, J = 6.8Hz, CH of i-Pr), 7.52 (1H, t, J = 8.1Hz,
benzene), 8.26 (2H, d, J = 8.1Hz, benzene), 8.69 (2H,
m, benzene and NH); FAB-MS m/z: 362 (M++1).
5.1.28. N-(3,4-Dimethyl-5-isopropyl-1,3-thiazol-2(3H)-
ylidene)-3-nitrobenzenesulfonamide (17e). 56% yield; mp
1
124–125ꢁC (diethyl ether–hexane). H NMR (DMSO-
5.1.24. N-(5-tert-Butyl-3,4-dimethyl-1,3-thiazol-2(3H)-
ylidene)-3-nitrobenzenesulfonamide (17a). To a solution
of 4 (8.49g, 23.9mmol) in tetrahydrofuran (150mL)
was added sodium hydride (60% dispersion in mineral
oil: 1.44g, 35.9mmol) and iodomethane (2.98mL,
47.8mmol) under ice-bath cooling. The solution was
warmed to room temperature and stirred for 12h. The
reaction mixture was poured into ice water and ex-
tracted with ethyl acetate. The organic layer was washed
with brine, and then dried over anhydrous sodium sul-
fate. The solvent was removed under reduced pressure
and the residue was purified by silica gel column chro-
matography (chloroform) and recrystallized from meth-
anol to give 17a (6.56g, 74%) as a yellow crystals. Mp
d6) d: 1.15 (6H, d, J = 6.9Hz, CH3 of i-Pr), 2.17 (3H,
s, 4-Me), 3.21 (1H, heptet, J = 6.9Hz, CH of i-Pr),
3.42 (3H, s, 3-Me), 7.85 (1H, dd, J = 7.8, 8.3Hz, benz-
ene), 8.26 (1H, ddd, J = 1.0, 1.9, 7.8Hz, benzene), 8.43
(1H, ddd, J = 1.0, 1.9, 8.3Hz, benzene), 8.49 (1H, t,
J = 1.9Hz, benzene); FAB-MS m/z: 356 (M++1).
Anal. Calcd for C14H17N3O4S2: C, 47.31; H, 4.82; N,
11.82; S, 18.04. Found: C, 47.27; H, 4.70; N, 11.88; S,
18.09.
5.1.29. N-(5-Acetyl-3,4-dimethyl-1,3-thiazol-2(3H)-ylid-
ene)-3-nitrobenzenesulfonamide (17f). 66% yield; mp
1
205–206ꢁC (acetonitrile). H NMR (DMSO-d6) d: 2.08
(3H, s, 4-Me), 2.48 (3H, s, CH3 ofAc), 2.58 (3H, s, 3-
Me), 7.88 (1H, t, J = 8.0Hz, benzene), 8.30 (1H, ddd,
J = 0.9, 1.8, 8.0Hz, benzene), 8.46 (1H, ddd, 0.9, 1.8,
8.0Hz, benzene), 8.51 (1H, t, J = 1.8Hz, benzene);
FAB-MS m/z: 356 (M++1). Anal. Calcd for
C13H13N3O5S2: C, 43.94; H, 3.69; N, 11.82; S, 18.05.
Found: C, 44.11; H, 3.60; N, 11.99; S, 17.85.
1
143–145ꢁC. H NMR (DMSO-d6) d: 1.32 (9H, s, CH3
of t-Bu), 2.12 (3H, s, 4-Me), 3.60 (3H, s, 3-Me), 7.84
(1H, dd, J = 7.9, 8.3Hz, benzene), 8.26 (1H, br d,
J = 7.9Hz, benzene), 8.41 (1H, dd, J = 2.5, 8.3Hz, ben-
zene), 8.49 (1H, t, J = 2.5Hz, benzene); FAB-MS m/z:
370 (M++1). Anal. Calcd for C15H19N3O4S2: C, 48.76;
H, 5.18; N, 11.37; S, 17.36. Found: C, 48.69; H, 5.25;
N, 11.31; S, 17.37.
5.1.30. N-(5-Isopropyl-3-methyl-1,3-thiazol-2(3H)-ylid-
ene)-3-nitrobenzenesulfonamide (17g). 76% yield; mp
150–151ꢁC (isopropanol). 1H NMR (DMSO-d6) d:
1.19 (6H, d, J = 6.9Hz, CH3 of i-Pr), 2.95 (1H, heptet,
J = 6.9Hz, CH of i-Pr), 3.45 (3H, s, 3-Me), 7.22 (1H,
s, thiazole), 7.86 (1H, d, J = 7.8, 8.3Hz, benzene), 8.26
(1H, br d, J = 7.8Hz, benzene), 8.43 (1H, ddd, J = 1.0,
1.9, 8.3Hz, benzene), 8.49 (1H, t, J = 1.9Hz, benzene);
FAB-MS m/z: 342 (M++1). Anal. Calcd for
C13H15N3O4S2: C, 45.73; H, 4.43; N, 12.31; S, 18.78.
Found: C, 45.66; H, 4.31; N, 12.30; S, 18.89.
The following compounds were obtained in the same
manner.
5.1.25.
N-(3,4-Dimethyl-1,3-thiazol-2(3H)-ylidene)-3-
nitrobenzenesulfonamide (17b). 80% yield; mp 199–
200ꢁC (H2O–acetonitrile), 1H NMR (DMSO-d6) d:
2.21 (3H, s, 4-Me), 3.42 (3H, s, 3-Me), 6.63 (1H, s, thi-
azole), 7.85 (1H, t, J = 7.8Hz, benzene), 8.25 (1H, br
d, J = 7.8Hz, benzene), 8.42 (1H, ddd, J = 1.0, 2.0,
8.3Hz, benzene), 8.49 (1H, dd, J = 1.5, 2.0Hz, benzene),
FAB-MS m/z: 313 (M+). Anal. Calcd for C11H11N3O4-
S2Æ0.3H2O: C, 41.45; H, 3.67; N, 13.18; S, 20.12. Found:
C, 41.44; H, 3.40; N, 13.36; S, 20.24.
5.1.31. N-(4-Isobutyl-5-isopropyl-3-methyl-1,3-thiazol-
2(3H)-ylidene)-3-nitrobenzenesulfonamide (17h). 41%
yield; mp 125–126ꢁC (isopropanol). 1H NMR
(DMSO-d6) d: 0.90 (6H, d, J = 6.3Hz, CH3 of i-Bu),
1.16 (6H, d, J = 6.8Hz, CH3 of i-Pr), 1.78 (1H, m, CH
of i-Bu), 2.48 (2H, d, J = 7.3Hz, CH2 of i-Bu), 3.12
(1H, heptet, J = 6.8Hz, CH of i-Pr), 3.42 (3H, s, 3-
Me), 7.86 (1H, dd, J = 7.8, 8.3Hz, benzene), 8.23 (1H,
br d, J = 7.8Hz, benzene), 8.42 (1H, ddd, J = 1.0, 2.0,
7.8Hz, benzene), 8.50 (1H, t, J = 2.0Hz, benzene);
5.1.26. N-(3,4,5-Trimethyl-1,3-thiazol-2(3H)-ylidene)-3-
nitrobenzenesulfonamide (17c). 88% yield; mp 156–
1
158ꢁC (diethyl ether–acetonitrile), H NMR (DMSO-
d6) d: 2.13 (3H, s, 4-Me), 2.16 (3H, s, 5-Me), 3.43 (3H,
s, 3-Me), 7.84 (1H, dd, J = 7.8, 8.3Hz, benzene), 8.24
(1H, br d, J = 7.8Hz, benzene), 8.42 (1H, ddd, J = 0.9,

6180
N. Masuda et al. / Bioorg. Med. Chem. 12 (2004) 6171–6182
FAB-MS m/z: 398 (M++1). Anal. Calcd for
C17H23N3O4S: C, 51.36; H, 5.83; N, 10.57; S, 16.13.
Found: C, 51.31; H, 5.82; N, 10.62; S, 15.96.
tion mixture was quenched with aqueous acetone and
evaporated. The residue was diluted with ethyl acetate
and washed with water and brine, then dried over anhydr-
ous sodium sulfate. The solvent was removed under
reduced pressure and the residue was purified by silica
gel column chromatography (chloroform–methanol)
and recrystallized from isopropanol. To the solution of
the product and triethylamine (1.39mL, 10.0mmol) in
dichloromethane (20mL) was added methanesulfonyl
chloride (0.77mL, 10.0mmol) under ice-bath cooling.
After the reaction mixture was stirred at the same tem-
perature for 30min, the solution was warmed to room
temperature and stirred for 36h. The reaction mixture
was evaporated and diluted with chloroform. The solu-
tion was washed with brine, and then dried over anhy-
drous sodium sulfate. The solvent was removed under
reduced pressure and the residue was purified by silica
gel column chromatography (ethyl acetate–hexane)
and recrystallized from ethanol to give 18 (459mg,
5.1.32.
N-(5-Isopropyl-3-methyl-4-trifluoromethyl-1,3-
thiazol-2(3H)-2-ylidene)-3-nitrobenzenesulfonamide (17i).
19% yield; mp 127–128ꢁC (ethyl acetate–hexane). 1H
NMR (CDCl3) d: 1.31 (6H, d, J = 7.0Hz, CH3 of i-
Pr), 3.53 (1H, m, CH of i-Pr), 3.58 (3H, br s, 3-Me),
7.70 (1H, t, J = 7.8Hz, benzene), 8.30 (1H, dt, J = 2.0,
7.8Hz, benzene), 8.39 (1H, m, benzene), 8.79 (1H, t,
J = 2.0Hz, benzene); FAB-MS m/z: 410 (M++1). Anal.
Calcd for C14H14F3N3O4S2: C, 41.07; H, 3.45; N,
10.26; S, 15.66; F, 13.92. Found: C, 41.02; H, 3.37; N,
10.23; S, 15.64; F, 13.83.
5.1.33. Methyl 5-isopropyl-3-methyl-2-{[(3-nitrophenyl)-
sulfonyl]imino}-2,3-dihydro-1,3-thiazole-4-carboxylate (17j).
43% yield; mp 120–121ꢁC (ethyl acetate–hexane). 1H
NMR (CDCl3) d: 1.29 (6H, d, J = 6.8Hz, CH3 of i-
Pr), 3.67 (3H, s, 3-Me), 3.68 (1H, heptet, J = 6.8Hz,
CH of i-Pr), 3.93 (3H, s, CH3 ofCOOMe), 7.88 (1H,
br t, J = 8.2Hz, benzene), 8.29 (1H, br d, J = 7.7Hz,
benzene), 8.37 (1H, dd, J = 2.0, 8.2Hz, benzene), 8.79
(1H, t, J = 2.0Hz, benzene); FAB-MS m/z: 400
(M++1). Anal. Calcd for C15H17N3O6S2: C, 45.10; H,
4.29; N, 10.52; S, 16.06. Found: C, 44.93; H, 4.10; N,
10.62; S, 16.09.
1
21%) as a yellow crystals. Mp 171–173ꢁC. H NMR
(DMSO-d6) d: 2.27 (3H, s, 4-Me), 3.44 (3H, s, 3-Me),
5.21 (1H, d, J = 10.8Hz, –CH@CH2), 5.33 (1H, d,
J = 17.0Hz, –CH@CH2), 6.89 (1H, dd, J = 10.8,
17.0Hz, –CH@CH2), 7.86 (1H, t, J = 8.3Hz, benzene),
8.28 (1H, br d, J = 7.8Hz, benzene), 8.43 (1H, dd,
J = 2.5, 7.8Hz, benzene), 8.50 (1H, t, J = 2.5Hz, ben-
zene); FAB-MS m/z: 340 (M++1). Anal. Calcd for
C13H13N3O4S2: C, 46.01; H, 3.86; N, 12.38; S, 18.90.
Found: C, 45.93; H, 3.87; N, 12.10; S, 18.46.
5.1.34.
N-(4-Chloro-5-isopropyl-3-methyl-1,3-thiazol-
2(3H)-ylidene)-3-nitrobenzenesulfonamide (17k). 52%
yield; mp 140–142ꢁC (diethyl ether). 1H NMR
(DMSO-d6) d: 1.20 (6H, d, J = 6.8Hz, CH3 of i-Pr),
3.18 (1H, heptet, J = 6.8Hz, CH of i-Pr), 3.47 (3H, s,
3-Me), 7.87 (1H, t, J = 7.8Hz, benzene), 8.28 (1H, br
d, J = 7.9Hz, benzene), 8.45 (1H, br d, J = 7.9Hz, benz-
ene), 8.50 (1H, br s, benzene); FAB-MS m/z: 376
(M++1). Anal. Calcd for C13H14N3O4S2Cl: C, 41.54;
H, 3.75; N, 11.18; S, 17.06; Cl, 9.43. Found: C, 41.89;
H, 3.74; N, 10.95; S, 16.85; Cl, 9.81.
5.1.37.
5-Isopropyl-3-methyl-2-{[(3-nitrophenyl)sulfo-
nyl]imino}-2,3-dihydro-1,3-thiazole-4-carboxamide (19).
To a solution of 17j (877mg, 2.19mmol) in tetra-
hydrofuran (10mL) was added saturated aqueous
ammonia solution (10mL) and heated to 120ꢁC in auto-
clave and stirred for 3h. The mixture was evaporated in
reduced pressure. To the residue was added thionyl chlo-
ride (1mL), N,N-dimethylformamide (0.15mL) and stir-
red at 70ꢁC for 1h. Thionyl chloride was removed under
reduced pressure and the residue was solved to chloro-
form (10mL). To saturated aqueous ammonia (10mL)
was added dropwise the solution under ice-bath cooling
and stirred at same temperature for 15min. The reaction
mixture was extracted with ethyl acetate. The organic
layer was washed with brine, and then dried over anhy-
drous sodium sulfate. The solvent was removed under
reduced pressure and the residue was recrystallized from
ethyl acetate–hexane to give 19 (547mg, 65%) as an or-
5.1.35.
N-(5-tert-Butyl-3-ethyl-4-methyl-1,3-thiazol-
2(3H)-ylidene)-3-nitrobenzenesulfonamide (17l). Com-
pound 17l was obtained in the same manner as described
in the synthesis of 17a with iodoethane instead ofiodo-
methane. 50% yield; mp 139–141ꢁC (diethyl ether–hex-
1
ane). H NMR (DMSO-d6) d: 1.12 (3H, t, J = 6.8Hz,
CH3 ofEt), 1.32 (9H, s, CH of t-Bu), 2.31 (3H, s, 4-
3
1
Me), 3.96 (2H, q, J = 6.8Hz, CH2 ofEt), 7.85 (1H, t,
J = 8.3Hz, benzene), 8.25 (1H, ddd, J = 1.0, 1.9,
8.3Hz, benzene), 8.42 (1H, dd, J = 1.0, 1.9, 8.3Hz, benz-
ene), 8.48 (1H, t, J = 1.9Hz, benzene); FAB-MS m/z:
384 (M++1). Anal. Calcd for C16H21N3O4S2: C, 50.11;
H, 5.52; N, 10.96; S, 16.72. Found: C, 50.06; H, 5.49;
N, 11.02; S, 16.81.
ange crystals. Mp 246–248ꢁC. H NMR (DMSO-d6) d:
1.20 (6H, d, J = 7.0Hz, CH3 of i-Pr), 3.31 (1H, m, CH
of i-Pr), 3.41 (3H, s, 3-Me), 7.87 (1H, t, J = 8.3Hz, ben-
zene), 8.15 (1H, s, NH2), 8.19 (1H, s, NH2), 8.28 (1H, br
d, J = 8.3Hz, benzene), 8.44 (1H, br d, J = 8.3Hz, ben-
zene), 8.50 (1H, m, benzene); FAB-MS m/z: 385
(M++1). Anal. Calcd for C14H16N4O5S2: C, 43.74; H,
4.20; N, 14.57; S, 16.68. Found: C, 43.68; H, 4.35; N,
14.34; S, 16.37.
5.1.36. N-(3,4-Dimethyl-5-vinyl-1,3-thiazol-2(3H)-ylid-
ene)-3-nitrobenzenesulfonamide (18). To a solution of
17f (1.78g, 5.01mmol) in methanol (50mL) was added
sodiumborohydride (0.38g, 10.0mmol) under ice-bath
cooling. The mixture was warmed to room temperature
and stirred for 2.5h. Additional sodiumborohydride
(0.19g, 5.0mmol) was added and stirred 12h. The reac-
5.1.38.
2(3H)-ylidene)-3-nitrobenzenesulfonamide (20). To
solution of 19 (268mg, 0.70mmol) in 1,2-dichloroethane
(5mL) was added phosphorous oxychloride (0.33mL,
3.50mmol) and N,N-dimethylformamide (0.10mL) un-
N-(4-Cyano-5-isopropyl-3-methyl-1,3-thiazol-
a

N. Masuda et al. / Bioorg. Med. Chem. 12 (2004) 6171–6182
6181
der ice-bath cooling. The mixture was heated to 70ꢁC
and stirred for 1h. The reaction mixture was pored into
ice water and extracted with ethyl acetate. The organic
layer was washed with brine, and then dried over anhy-
drous sodium sulfate. The solvent was removed under
reduced pressure and the residue was recrystallized from
ethyl acetate–hexane to give 20 (180mg, 70%) as an or-
ange crystals. Mp 136–137ꢁC. 1H NMR (CDCl3) d: 1.37
(6H, d, J = 7.0Hz, CH3 of i-Pr), 3.38 (1H, heptet,
J = 7.0Hz, CH of i-Pr), 3.59 (3H, s, 3-Me), 7.70 (1H,
t, J = 7.9Hz, benzene), 8.27 (1H, br d, J = 7.9Hz, ben-
zene), 8.40 (1H, ddd, J = 2.0, 2.2, 7.9Hz, benzene),
8.76 (1H, t, J = 2.0Hz, benzene); FAB-MS m/z: 367
(M++1). Anal. Calcd for C14H14N4O4S2: C, 45.89; H,
3.85; N, 15.29; S, 17.50. Found: C, 45.70; H, 3.65; N,
15.32; S, 17.24.
UTX81 and purified by the scheme described by Saitoh
et al.21 In vitro RT assays were conducted according to
the previously described method with the following
modifications.22 Test compounds and 0.01unit ofrec-
ombinant HIV-1 RT enzymes (either wild type or mu-
tant) were incubated in a reaction mixture (50lL)
containing 50mM Tris–HCl (pH8.4), 100mM KCl,
10mM MgCl2, 0.1% Triton X-100, 2mM dithiothreitol,
0.01 OD260 ofpoly(rC)/oligo(dG) 12–18, and 1lCi of
[10,20-3H]dGTP (33Ci/mmol) at 37ꢁC for 1h. The reac-
tion was stopped with 200lL of5% cold trichloroacetic
acid. The precipitated materials were analyzed for radio
activity using a scintillation counter (Aloka Co., Ltd,
Tokyo, Japan).
5.2.4. X-ray crystal structure analysis of 17a. A single
crystal of 17a was obtained by recrystallization from
MeOH. Crystal data for 17a: C15H19N3O4S2;
Mr = 369.45, colorless plate, crystal size 0.45 · 0.18 ·
5.2. Pharmacology
5.2.1. Cells and viruses. MT-4 cells18 and two strains of
HIV-1, HIV-1IIIB and HIV-1IIIB-R, were used for the
anti-HIV-1 assays. HIV-1IIIB-R, which has a single
amino acid change (Y181C) in its RT, is established
by serial passages in cell cultures in the presence ofesca-
lating concentrations ofMKC-442. 19 HIV-1IIIB-R is also
resistant to nevirapine. MT-4 cells were grown and
maintained in RPMI-1640 medium supplemented with
10% heat-inactivated fetal bovine serum (FBS), penicil-
lin G (100units/mL), and gentamicin (20mg/mL). MT-
4 cells and HIV-1IIIB were obtained from Rational Drug
Design Laboratories (Fukushima, Japan).
0.02mm, triclinic, space group P-1, a = 10.1641(6)A,
˚
˚
˚
b = 10.7000(6)A, c = 9.6632(5)A, a = 90.408(5)ꢁ, b =
3
˚
106.252(5)ꢁ, c = 63.298(4)ꢁ, V = 892.67(9)A , Z = 2,
Dcalc = 1.374g/cm3,
F(000) = 388,
l(CuKa) =
29.21cmꢀ1. Data were collected on a RIGAKU AFC5R
diffractometer at 298K. Lattice constants were obtained
from a least-squares refinement using the setting angles
of25 reflections careuflly centered in the range of
55.34ꢁ < 2h < 62.31ꢁ. The structure of 17a was solved
by direct methods using the SIR92 program.23 The data
were corrected for Lorentz and polarization effects. An
experimental absorption correction was also applied.
All hydrogen atoms were located from difference Fou-
rier synthesis. Full-matrix least-squares refinement was
carried out and converged with a final calculated R-fac-
tor [I > 3r(I)] of0.043 ( Rw = 0.062) and a goodness-of-
fit of1.189.
5.2.2. Anti-HIV-1 assay. Determination ofthe antiviral
activity ofthe test compounds against HIV-1 IIIB replica-
tion was based on the inhibition ofvirus-induced cyto-
pathicity in MT-4 cells. Briefly, MT-4 cells were
suspended in culture medium at 1 · 105 cells/mL and in-
fected with virus at a multiplicity of infection (MOI) of
0.02. Immediately after virus infection, the cell suspen-
sion (100lL) was brought into each well ofa flat-bot-
tomed microtiter tray containing various concen-
trations ofthe test compounds. After a 5-day incubation
at 37ꢁC, the number ofviable cells was determined by
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) method.20 The HTS ofour compound
5.2.5. Docking study. Docking studies for compound 17a
with HIV reverse transcriptase24 (RT) nonnucleoside
binding sites were performed using the GOLD pro-
gram.25 Ten independent genetic algorithms (GA) in
which a maximum number of100,000GA operations
were performed on a single population of 100 individu-
als, were calculated. Operator weights for crossover,
mutation, and migration were set to 95, 95, and 10,
respectively.
library was also performed using the MTT assay against
19
HIV-1IIIB-R
oftest compounds were expressed as EC
.
The anti-HIV-1 activity and cytotoxicity
and CC50,
50
respectively. EC50 is the concentration ofa test com-
pound that was able to suppress HIV-1 replication by
50%. CC50 is the concentration ofa test sample that
reduced viable cell number by 50% in mock-infected
cells. The therapeutic index (TI) is the ratio ofCC to
50
EC50.
Acknowledgements
We wish to acknowledge helpful discussion with Dr.
Susumu Igarashi and Dr. Hirokazu Kubota. We also
thank members ofthe Division ofAnalytical Research
for performing instrumental analysis.
5.2.3. In vitro RT inhibition assay. A expression plas-
mide, pG280, which encodes HIV-1 RT proteins as
LacZ fusion proteins were used for the expression of
WT RT and mutated RTs.21 The single amino acid-sub-
stituted RTs (K103N RT and Y181C RT) were con-
structed using pG280 from a QuikchangeTM Site-
Directed Mutagenesis Kit (Stratagene, La Jolla, CA).
Recombinant RT enzymes were expressed in E. coli.
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